Solving the insoluble (and watching them dance)

By Jeremy Berg

I recently have started to work on the next edition of “Biochemistry,” the textbook first written by Lubert Stryer. The initiation of the revision process is always a bit daunting, but it is a great occasion to take stock of progress across the entire field of biochemistry. In my survey, four facets stood out: newly appreciated roles for RNA; an increased interest in the importance of metabolism; the ever-growing knowledge of the vastness of the microbial world, including the human microbiome; and the structures and mechanisms of action of membrane proteins. I will focus on the last topic here.

Membrane proteins, of course, play hugely important roles in almost all aspects of biochemistry and molecular biology as receptors, ion channels, other transporters and enzymes that act on lipid substrates, among others. Furthermore, proteins found through genetic studies often can be identified as membrane proteins by virtue of characteristic stretches of relatively hydrophobic amino acids in their deduced primary structures. Progress toward understanding the structures and mechanisms of these proteins, despite their importance, has been relatively slow until recently for several reasons.

First, almost by definition, membrane proteins are insoluble in aqueous buffers. Purification techniques that are so effective for most soluble proteins need to be modified for membrane proteins. Membrane proteins must be solubilized through the use of detergents or other amphipathic molecules, and the micelles formed are the actual subjects of purification.

Second, most membrane proteins are quite conformationally flexible and dynamic. This is not simply a consequence of the fact that they must be removed from their natural lipid-based environments for purification. Many depend on substantial conformational changes for their function, as in inactive versus activated forms of a receptor or open and closed states of a channel. This makes the purification of a conformationally homogenous sample, not just a pure covalent polypeptide chain, additionally challenging.

Finally, many membrane proteins, particularly those from human beings and other eukaryotes, can be quite complex, with several domains or multiple subunits.

The structural biology of membrane proteins was launched with the low-resolution determination of the structure of bacteriorhodopsin in the mid-1970s by electron microscopy and the determination of the bacterial photosynthetic reaction center in the early 1980s. With the development of molecular biology techniques for protein expression and engineering and the cloning of the genes for many key membrane proteins, the possibilities seemed limitless. Yet advances came quite slowly. This was due partially to the challenges noted above. The availability of a range of highly purified detergents was required to examine empirically different purification and crystallization protocols to find the most effective ones. The use of appropriate ligands or antibody fragments facilitated locking membrane proteins into single conformational states in some cases. Finally, in some cases, prokaryotic sequences that represented simpler versions of eukaryotic proteins of interest could be identified.

Another problem emerged related to financial support for membrane-protein structural biology. Agencies in Europe and organizations including the Howard Hughes Medical Institute displayed long-term interest in the field. However, the conservative nature of grant-review panels presented barriers, as the purification and crystallization of membrane proteins is very challenging and can be quite expensive. Reviewers were confronted with the task of comparing applications describing structural studies of interesting soluble proteins for which purified material and crystals already were available with those applications proposing the purification and crystallization of membrane proteins. Almost invariably, the proposals with crystals in hand won out. The National Institutes of Health tried many approaches to address this issue, including program announcements clearly articulating its interest in facilitating membrane-protein structural biology but not setting aside funds specifically for this purpose.

This approach had only limited success, so the NIH set aside funds for membrane-protein structural biology as components of its Roadmap and the National Institute of General Medical Sciences’ Protein Structure Initiative. Many outstanding proposals were submitted, and considerable progress was made both on general methods and on specific membrane-protein structures.

Another NIH investment also played an important role. During the agency’s budget doubling, NIGMS and the National Cancer Institute committed to building new synchrotron beamlines at the Advanced Photon Source at Argonne National Laboratory. One of these is capable of producing a very intense beam with dimensions of less than 10 microns. This allows examination of crystals too small to be useful at other sources and the scanning of larger crystals to find small regions that are well-ordered for data collection.

What progress has been made through these investments? One of the most spectacular successes was the determination of the structures of G-proteincoupled receptors including the β2-adrenergic receptor that was the subject of my column in December. The research that led to this structure was supported by the NIH through a variety of mechanisms, including the Roadmap and Protein Structure Initiative programs, and some key data sets were collected on the microfocus beamline at Argonne. More general data about progress on membrane-protein structural biology is compiled at various databases, including the Membrane Proteins of Known 3D Structure, which tracks both the total number of membrane-protein coordinate sets and the number of unique membrane-protein structures – that is, those with truly distinct polypeptide composition (i.e., not separately counting structures with different ligands bound or simple mutations). The number of unique structures grew from one in 1985 to five in 1993 to 83 in 2003 and 415 in 2013 (to date). Included in the list are representatives from almost all major classes of membrane proteins, including receptors, ligand- and voltage-gated ion channels, ion pumps, transporters of various classes, and a range of membrane-bound enzymes. Membrane proteins represent approximately half of the targets of drugs, and the structures of many of these have been solved.

However, for most membrane proteins, a single structure does not tell the whole story because, as noted above, most membrane proteins undergo large conformational changes in the course of performing their functions. What is particularly exciting is the availability of structures for a given protein in a range of conformational states, often captured through the use of different bound ligands: receptors in their inactive and activated states, ion channels in several distinct closed and open forms, ion pumps in states throughout their pumping cycles, transporters open to either side of the membrane. Many of these structures reveal dramatic domain motions and other conformational changes. These structures can be integrated to construct complete approximations of full functional cycles either by interpolating between structures or by more sophisticated molecular dynamics calculations (see the Membrane Protein Structural Dynamics Gateway and the selected publications of D.E. Shaw Research). The depictions of these molecular dances are quite aesthetically appealing, imbuing the molecules with lifelike features as they twist and jiggle into new shapes. More importantly, these simulations can provide additional insights into mechanism and can suggest incisive experiments.

Lubert Stryer. Photo courtesy of Wikimedia Commons.

As I prepare myself for the beginning of a new textbook revision, I go back and reread to the first edition of Stryer’s “Biochemistry,” published in 1975. I first learned biochemistry from that wonderful book. I am always struck by how much progress has been made. Many of the topics that were hinted at but covered only briefly are now much more fully understood. For example, the first edition contains many pictures of crystals of proteins that had been grown but for which no structure was yet available. These were clearly included as promises for things to come. Moreover, it feels as if Dr. Stryer had to work to find topics for which sufficient information was available for a reasonable discussion. This is very different from the experience of writing a biochemistry text today. My desk and computer drives are littered with papers to be considered for inclusion, but the pile of topics that are fascinating and important but for which there is insufficient space is much larger than the one for the topics that make it in. As is often the case, the more we know, the more we realize how much we don’t know.

Jeremy Berg (jberg@pitt.edu) is the associate senior vice-chancellor for science strategy and planning in the health sciences and a professor in the computational and systems biology department at the University of Pittsburgh.